Creation of a three-dimensional spherical fluorescence spot for super-resolution microscopy using a two-color annular hybrid wave plate.

We fabricate a two-color annular hybrid wave plate (TAHWP) for fluorescence depletion super-resolution microscopy by combining quartz plates. Inserting the TAHWP into a commercial laser-scanning microscope together with a quarter-wave plate creates a three-dimensional spherical fluorescence spot smaller than the diffraction limit. Since a simple experimental setup easily provides a spot with a volume of ∼100 nm3, the TAHWP can improve the performance of various microspectroscopic devices.

Fabrication of optical multilayer for two-color phase plate in super-resolution microscope.

In super-resolution microscopy based on fluorescence depletion, the two-color phase plate (TPP) is an indispensable optical element, which can independently control the phase shifts for two beams of different color, i.e., the pump and erase beams. By controlling a phase shift of the erase beam through the TPP, the erase beam can be modulated into a doughnut shape, while the pump beam maintains the initial Gaussian shape. To obtain a reliable optical multiplayer (ML) for the TPP, we designed a ML with only two optical layers by performing numerical optimization. The measured phase shifts generated by the fabricated ML using interferometry correspond to the design values. The beam profiles in the focal plane are also consistent with theoretical results. Although the fabricated ML consists of only two optical layers, the ML can provide a suitable phase modulation function for the TPP in a practical super-resolution microscope.

New design method for a phase plate in super-resolution fluorescence microscopy.

The performance of a super-resolution fluorescence depletion microscope system depends crucially on the precise alignment of the pump and erase beams with the axis of the focusing objective. Here, we propose a new design method for a two-color spiral phase plate with a single-layer structure (S2SPP), and we experimentally investigate the image properties given by the phase plate. In spite of its simple structure, the plate can provide a super-resolution image with a spatial resolution better than 70 nm. Beside eliminating alignment problems and yielding a compact setup, the simplicity of fabrication of the S2SPP makes our proposed method very suitable for commercial microscope systems.

Two-photon absorption in rhodamine 6G occurring in concert with fluorescence depletion.

Using two-color fluorescence dip spectroscopy, we observed two-photon absorption processes (TPA) in Rhodamine 6G occurring in concert with fluorescence depletion processes (FD). A nano-second pulse erase beam in the near-infrared region was applied to induce TPA together with FD. It was found that the fluorescence intensity strongly depends on the photon-flux of the erase beam and that the effective TPA cross-section for the nano-second laser pulse is smaller than that for a femto-second laser pulse. This phenomenon can be interpreted as resulting from FD induced by a multiple-excitation process.

Laguerre-Gaussian radial Hilbert transform for edge-enhancement Fourier transform x-ray microscopy.

An efficient technique to achieve isotropic edge enhancement in optics involves applying a radial Hilbert transform on the object spectrum. Here we demonstrate a simple setup for isotropic edge-enhancement in soft-x- ray microscopy, using a single diffractive Laguerre-Gaussian zone plate (LGZP) for radial Hilbert transform. Since the LGZP acts as a beam-splitter, diffraction efficiency problems usually associated with x-ray microscopy optics are not present in this system. As numerically demonstrated, the setup can detect optical path differences as small as lambda/50 with high contrast.

Investigation of the center intensity of first- and second-order Laguerre-Gaussian beams with linear and circular polarization.

The vectorial Debye integral shows that tightly focused Laguerre-Gaussian (LG) beams have a residual intensity at the focal point for linear polarization, for a topological charge of m=1 and 2. We measured the shapes of linearly and circularly polarized LG beams and found that a central intensity appeared at m=1 and 2 for linear and right-handed circular polarization, however, it is completely canceled for left-handed circular polarization. In general, when the orbital angular momentum of the LG beam is parallel to the spin angular momentum of the photons, zero intensity is always achieved at the focus.

Measurement of contrast transfer function in super-resolution microscopy using two-color fluorescence dip spectroscopy.

The contrast transfer function (CTF) of super-resolution microscopy was quantitatively investigated using a fluorescent scale. The scale has minute fluorescent line patterns, finer than 100 nm, and is suitable for measuring CTF in super-resolution microscopy. The measured CTF shows that super-resolution microscopy can indeed improve the optical properties of fluorescent images and enable us to observe a structure with the spatial resolution overcoming the diffraction limit. From the CTF, it has been found that super-resolution microcopy can resolve a 100 nm line-and-space pattern and provides a contrast of 10%. The CTF corresponds to a PSF with a full-width at half-maximum (FWHM) of 130 nm. An evaluation using a 100 nmphi fluorescent bead consistently supports the results given by the CTF for super-resolution microscopy.

Two-point separation in far-field super-resolution fluorescence microscopy based on two-color fluorescence dip spectroscopy, Part I: Experimental evaluation.

The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.

Two-point-separation in super-resolution fluorescence microscope based on up-conversion fluorescence depletion technique.

Pronounced separation (750 nm) between two individual fluorescence spots in a novel super-resolution microscopy based on a two-color up-conversion fluorescence depletion technique has been investigated. This microscopy has the potential to achieve a spatial resolution (<300nm) of 1/2 the diffraction limit.